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D103A
-
site-directed mutagenesis of the catalytic residue, the mutant shows 5000fold reduced activity compared tot he wild-type enzyme
D38A
-
the catalytic turnover number is 140fold less than that for the wild-type
D38A/D99N
-
no detectable activity
D38A/Y14F
-
no detectable activity
D38A/Y55F
-
no detectable activity
D40N
-
site-directed mutagenesis, the mutation mimics the protonated aspartate found in the intermediate and equilenin complexes and leads to tighter binding of phenolate and other intermediate analogs
D99A
-
secondary kinetic isotope effects similar to wild-type. Mutation does not significantly decrease the contribution of coupled motion/hydrogen tunneling to the enzymatic reaction
D99L
the mutant shows about 10000fold decreased kcat compared to the wild type enzyme
F116W
the turnover-number for 5-androstene-3,17-dione is lowered 4.42fold, the KM-value is 3.1fold lower than the Km-value of the wild-type enzyme
Y14F/D99L
the mutant shows 1000000fold decreased kcat compared to the wild type enzyme
Y16F
-
site-directed mutagenesis of the catalytic residue, the mutant shows 50000fold reduced activity compared tot he wild-type enzyme
Y55F/Y88F
-
replacement of Tyr14 by 3-fluorotyrosine in the Y55,88F modified form of the isomerase results in a 4-fold decrease in turnover number
Y57F/D40N
-
site-directed mutagenesis, 3,4,5-trifluorophenol bind as ionized phenolate to KSI containing the Y57F mutation
D241N
-
mutant enzyme nearly has full isomerase activity, substantial decrease in 3beta-hydroxysteroid dehydrogenase activity. Mutant enzyme has a basal isomerase activity in the absence of coenzyme that is 10% of the NADH-stimulated turnover number
D257L
-
complete lack of isomerase activity in absence of coenzyme, substantial decrease in 3beta-hydroxysteroid dehydrogenase activity
D258L
-
complete lack of isomerase activity in absence of coenzyme, substantial decrease in 3beta-hydroxysteroid dehydrogenase activity
D265N
-
complete lack of isomerase activity in absence of coenzyme, substantial decrease in 3beta-hydroxysteroid dehydrogenase activity
D36A/K37R
-
mutation shifts the cofactor preference of both 3-beta-hydroxysteroid dehydrogenase and isomerase from NAD(H) to NADP(H)
DELTA283-310
-
the molecular weight of the subunit is 38800 Da compared to 42000 Da for the wild-type enzyme
E126L
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
H156Y
site-directed mutagenesis, leads to destabilization of interactions at the dimer interface and a dramatic increase in the substrate Km and inhibitor Ki values of 3beta-HSD isozyme 1 to equal those measured for 3beta-HSD isozyme 2
H232A
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
K158Q
site-directed mutagenesis, results in the complete loss of dehydrogenase activity and a reduction in isomerase activity, probably due to partial destabilization of cofactor binding, in which the catalytic triad is actively involved, and associated substrate binding
M187T
-
increase in affinity for NADH as an allosteric activator
N100A
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
N100S
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
N323L
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
Q105M
site-directed mutagenesis, leads to destabilization of interactions at the dimer interface and a dramatic increase in the substrate Km and inhibitor Ki values of 3beta-HSD isozyme 1 to equal those measured for 3beta-HSD isozyme 2
S124A
site-directed mutagenesis, results in the complete loss of dehydrogenase activity and a reduction in isomerase activity, probably due to partial destabilization of cofactor binding, in which the catalytic triad is actively involved, and associated substrate binding
S124T
-
11fold decrease in affinity to substrate
S322A
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
Y154F
site-directed mutagenesis, results in the complete loss of dehydrogenase activity and a reduction in isomerase activity, probably due to partial destabilization of cofactor binding, in which the catalytic triad is actively involved, and associated substrate binding
D103N/D40N
site-directed mutagenesis, 3,4,5-trifluorophenol bind as ionized phenolate to KSI containing the D103N mutation
D99E
-
turnover-number for 5-androstene-3,17-dione is 68.1% of the turnover number of the wild-type enzyme, the Km-value is 1.22fold higher than the Km-value of the wild-type enzyme
E118A
-
the free-energy change for unfolding in the absence of urea at 25°C is decreased by about 3.9 kcal/mol compared to wild-type value. Mutation increasex the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 4.46 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 33% of that of the wild-type enzyme, the Km-value is 348% of that of the wild-type enzyme
E118A/N120A
-
the free-energy change for unfolding in the absence of urea at 25°C is decreased by about 9.5 kcal/mol compared to wild-type value. Mutation increases the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 3.89 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 6% of that of the wild-type enzyme, the Km-value is 523% of that of the wild-type enzyme
F54A
-
turnover number for 5-androstene-3,17-dione is 17.1% of the turnover number for the wild-type enzyme, turnover number for 5(10)-estrene-3,17-dione is 15.3% of the turnover number of the wild-type enzyme,the KM-value for 5-androstene-3,17-dione is 116% of the KM-value of the wild-type enzyme, the KM-value for 5(10)-estrene-3,17-dione is 233% of the KM-value of the wild-type enzyme
F54L
-
turnover number for 5-androstene-3,17-dione is 41.9% of the turnover number of the wild-type enzyme, turnover number for 5(10)-estrene-3,17-dione is 38.7% of the turnover number of the wild-type enzyme, the KM-value for 5-androstene-3,17-dione is 154% of the KM-value of the wild-type enzyme, the KM-value for 5(10)-estrene-3,17-dione is 89.3% of the KM-value of the wild-type enzyme
F82A
-
turnover number for 5-androstene-3,17-dione is 2.5% of the turnover number of the wild-type enzyme, turnover number for 5(10)-estrene-3,17-dione is 2.3% of the turnover number of the wild-type enzyme,the KM-value for 5-androstene-3,17-dione is 29.9% of the KM-value of the wild-type enzyme, the KM-value for 5(10)-estrene-3,17-dione is 29.8% of the KM-value of the wild-type enzyme
F82L
-
turnover number for 5-androstene-3,17-dione is 12% of the turnover number of the wild-type enzyme, turnover number for 5(10)-estrene-3,17-dione is 67.6% of the turnover number of the wild-type enzyme, the KM-value for 5-androstene-3,17-dione is 108% of the KM-value of the wild-type enzyme, the KM-value for 5(10)-estrene-3,17-dione is 130% of the KM-value of the wild-type enzyme
L125A/V127A
-
mutation in small exterior hydrophobic cluster, decrease in conformational stability
L125F/V127F
-
mutation in small exterior hydrophobic cluster, slight increase in stability
N120A
-
the free-energy change for unfolding in the absence of urea at 25°C is decreased by about 7.8 kcal/mol compared to wild-type value. Mutation increasex the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 3.95 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 17% of that of the wild-type enzyme, the Km-value is 516% of that of the wild-type enzyme
R72A
-
the free-energy change for unfolding in the absence of urea at 25°C is decreased by about 3.8 kcal/mol compared to wild-type value. Mutation increasex the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 4.74 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 23% of that of the wild-type enzyme
W116A
-
turnover number for 5-androstene-3,17-dione is 1.5% of the turnover number of the wild-type enzyme, turnover number for 5(10)-estrene-3,17-dione is below 12%% of the turnover number of the wild-type enzyme, the KM-value for 5-androstene-3,17-dione is 348% of the KM-value of the wild-type enzyme
W116F
-
turnover number for 5-androstene-3,17-dione is 22.4% of the turnover number of the wild-type enzyme, the KM-value for 5(10)-estrene-3,17-dione is 299% of the KM-value of the wild-type enzyme
W116Y
-
turnover number for 5-androstene-3,17-dione is 21% of the turnover number of the wild-type enzyme
W92A
-
mutation in small exterior hydrophobic cluster, decrease in conformational stability. Crystallization data
W92A/L125A/V127A
-
mutation in small exterior hydrophobic cluster, decrease in conformational stability
Y115F
the mutant shows about 68% activity compared to the wild type enzyme
Y14F/D99E
-
turnover-number for 5-androstene-3,17-dione is less than 1% of the turnover number of the wild-type enzyme, the Km-value is 1.05fold lower than the Km-value of the wild-type enzyme
Y14F/D99N
-
turnover-number for 5-androstene-3,17-dione is less than 1% of the turnover number of the wild-type enzyme, the Km-value is 1.06fold lower than the Km-value of the wild-type enzyme
Y14F/Y30F
-
the turnover-number for 5-androstene-3,17-dione is about 1% of that of the wild-type enzyme, the Km-value is 1.57fold higher than that of the wild-type enzyme
Y14F/Y30F/Y55F
-
the turnover-number for 5-androstene-3,17-dione is about 2.1% of that of the wild-type enzyme, the Km-value is comparable to the Km-value of the wild-type enzyme. The hydrogen bond between Asp99 Odelta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by additional Y30F/Y55F mutations. The improvement in the catalytic activity of Y14F by the additional Y30f/Y55F mutation is due to the changes in the structural integrity at the catalytic site and the resulting restoration of the proton-transfer mechanism in Y14F/Y30F/Y55F
Y14F/Y30F/Y55F/D99L
-
the turnover-number for 5-androstene-3,17-dione is less than 1.2% of that of the wild-type enzyme, the Km-value is 1.8fold higher than that of the wild-type enzyme
Y14F/Y30F/Y55F/D99N
-
the turnover-number for 5-androstene-3,17-dione is less than 1% of that of the wild-type enzyme, the Km-value is 1.14fold higher than that of the wild-type enzyme
Y14F/Y55F
-
the turnover-number for 5-androstene-3,17-dione is about 1.2% of that of the wild-type enzyme, the Km-value is 1.75fold higher than that of the wild-type enzyme
Y16F/Y32F/Y57F
the number of water molecules directly hydrogen bonded to the ligand oxygen is one in the Y16F/Y32F/Y57F mutant
Y16S
the number of water molecules directly hydrogen bonded to the ligand oxygen is approximately two in the Y16S mutant
Y30F/Y55F/Y115F
the mutant shows about 57% activity compared to the wild type enzyme
Y57S
-
the mutation causes a large decrease in the catalytic performance of the enzyme
D38E
compared to wild-type, 200fold reduction in kcat-value. Analysis of thermodynamic parameters
D38E
-
the mutant gives similar free energies to the native enzyme, with catalytic constants approximately 200-300times less than in wild type enzyme
Y14F
-
secondary kinetic isotope effects similar to wild-type. Mutation does not significantly decrease the contribution of coupled motion/hydrogen tunneling to the enzymatic reaction
Y14F
-
study on backbone dynamics in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate. Mutation induces a substantial decrease in the order parameters in free enzyme, indicating that the backbone structures become significantly mobile by mutation, while the chemical shift analysis indicates that the structural perturbations are more profound than those of wild-type upon 19-nortestosterone hemisuccinate binding. In the 19-nortestosterone hemisuccinate complexed mutant, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remain flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in the mutant enzyme become even greater than those of wild-type
Y14F
the mutant shows 100000fold decreased kcat compared to the wild type enzyme
D40N
-
1484555fold decrease in turnover number, 4.46fold decrease in KM-value as compared to wild-type enzyme
D40N
-
site-directed mutagenesis, the mutation mimics the protonated aspartate found in the intermediate and equilenin complexes and leads to tighter binding of phenolate and other intermediate analogs
D40N
-
inhibition compared to wild-type enzyme, overview
D40N
-
the mutation does not perturb the gross tertiary and secondary structure of the enzyme The D40N mutant mimics the charge distribution of the enzyme active site in the intermediate state (protonated base), and is not the active form of the enzyme
D99L
-
the turnover number for 5-androstene-3,17-dione is 96.5fold lower than that of the wild-type enzyme, the KM-value is 1.9fold lower than that of the wild-type enzyme. Mutation results in a loss of conformational stability of 3.8 kcal/mol, at 25°C, pH 7.0
D99L
-
turnover-number for 5-androstene-3,17-dione is 125fold lower than the turnover number of the wild-type enzyme, the Km-value is 1.95fold lower than the Km-value of the wild-type enzyme
Y14F
-
mutation results in a loss of conformational stability of 4.4 kcal/mol, at 25°C, pH 7.0
Y14F
-
the hydrogen bond between Asp99 Odelta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by additional Y30F/Y55F mutations
Y14F
-
turnover-number for 5-androstene-3,17-dione is less than 1% of the turnover number of the wild-type enzyme, the Km-value is 1.87fold lower than the Km-value of the wild-type enzyme
Y14F
the mutant shows almost no activity compared to the wild type enzyme
Y14F/D99L
-
turnover-number for 5-androstene-3,17-dione is less than 1% of the turnover number of the wild-type enzyme, the Km-value is 1.95fold higher than the Km-value of the wild-type enzyme
Y14F/D99L
-
partly additive effect of mutations for both, catalysis and stability, increase in hydrophobic interaction while disrupting the hydrogen bond network, crystallization data
Y16F
-
2009fold decrease in turnover number, 3.5fold decrease in KM-value as compared to wild-type enzyme
Y16F
the number of water molecules directly hydrogen bonded to the ligand oxygen is one in the Y16F mutant
Y30F
-
the turnover number for 5-androstene-3,17-dione is 1.2fold lower than that of the wild-type enzyme, the KM-value is 1.1fold lower than that of the wild-type enzyme
Y30F
the mutant shows about 84% activity compared to the wild type enzyme
Y30F/D99L
-
the turnover number for 5-androstene-3,17-dione is 521.6fold lower than that of the wild-type enzyme, the KM-value is 1.47fold lower than that of the wild-type enzyme. Mutation results in a loss of conformational stability of 6.5 kcal/mol, at 25°C, pH 7.0
Y30F/D99L
-
disruption of hydrogen bond network, crystallization data
Y30F/Y55F
-
the turnover number for 5-androstene-3,17-dione is 1.99fold lower than that of the wild-type enzyme, the KM-value is comparable to that of the wild-type enzyme
Y30F/Y55F
the mutant shows about 50% activity compared to the wild type enzyme
Y55F
-
the turnover number for 5-androstene-3,17-dione is 6fold lower than that of the wild-type enzyme, the KM-value is 2.17fold lower than that of the wild-type enzyme. mutation results in a loss of conformational stability of 3.5 kcal/mol, at 25°C, pH 7.0. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30
Y55F
the mutant shows about 17% activity compared to the wild type enzyme
Y55F/D99L
-
the turnover number for 5-androstene-3,17-dione is 17692fold lower than that of the wild-type enzyme, the KM-value is 1.24fold lower than that of the wild-type enzyme. Mutation results in a loss of conformational stability of 7.9 kcal/mol, at 25°C, pH 7.0
Y55F/D99L
-
disruption of hydrogen bond network, crystallization data